Compositions and methods for preserving insulin-producing cells and insulin production and treating diabetes

ABSTRACT

Nuclear Transport Modifiers such as cSN50 and cSN50.1, afford in vivo islet protection following a 2-day course of intense treatment in autoimmune diabetes-prone, non-obese diabetic (NOD) mice, a widely used model of Type 1 diabetes (T1D), which resulted in a diabetes-free state for one year without apparent toxicity and the need to use insulin. cSN50 precipitously reduces the accumulation of islet-destructive autoreactive lymphocytes while enhancing activation-induced cell death of T and B lymphocytes derived from NOD mice. cSN50 attenuated pro-inflammatory cytokine and chemokine production in immune cells in this model of human T1D. cSN50 also provides cytoprotection of beta cells, therefore preserving residual insulin-producing capacity. Because intracellular delivery of a Nuclear Transport Modifier peptide such as cSN50 and cSN50.1 can result in lowering of blood glucose levels and may reducing insulin resistance, the compositions, methods and cells described herein can also be used for treating Type 2 diabetes (T2D).

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional application No.61/544,018, filed Oct. 6, 2011, which is incorporated herein byreference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under HL 5 P01 HL068744awarded by the National Institutes of Health/National Heart, Lung, andBlood Institute and under 1F32DK083161 and 5 K08 DK 090146 awarded bythe National Institutes of Health/National Institute of Diabetes,Digestive and Kidney Diseases. The government has certain rights in theinvention.

FIELD

The field of the invention is endocrinology, immunology, and cellbiology.

BACKGROUND

Insulin-dependent Type 1 diabetes (T1D), also known as Juvenile Diabetesor Insulin-Dependent Diabetes Mellitus, is a devastating autoimmunedisease that destroys beta cells within the pancreatic islets andafflicts over 10 million people worldwide. Their autoimmune process isknown to lead to hyperlipidemia and accelerated atherosclerosis. Thesepatients face life-long risks for blindness, cardiovascular and renaldiseases, and complications of insulin treatment. Increasing evidenceregarding the pathomechanism of T1D indicates that islets are destroyedby the relentless attack by autoreactive immune cells evolving from anaberrant action of the innate, in addition to adaptive, immune systemthat produces islet-toxic cytokines, chemokines, and other effectors ofislet inflammation.

T1D results from the progressive destruction of insulin-producing betacells in pancreatic islets caused by pro-inflammatory and pro-apoptoticeffectors of innate and adaptive immunity. Extraordinary advances withinsulin monotherapy and understanding of the critical role of theadaptive immune system in the T1D pathomechanism have not translated todiabetes reversal. Patients remain at risk for the serious complicationsinherent to the autoimmune and metabolic derangements in T1D. Patientswith end-stage diabetic nephropathy can receive simultaneouskidney-pancreas (SPK) transplants. Secondly, T1D patients who developedend-stage diabetic nephropathy and received a successful kidneytransplant are potentially eligible for pancreas-after-kidney (PAK)transplantation. Thirdly, T1D patients with normal renal function albeitwith difficult to control insulin therapy can be treated with pancreastransplant alone (PTA). These approaches have a limited success ratesalthough the positive outcome of SPK and PAK transplants includesdecreasing or reversing diabetic neuropathy (Jamiolkowski R M et al.Yale Journal of Biology and Medicine 85 (2012), pp. 37-43). Islettransplantation poses less risk than major organ transplant surgery.However, the risk of immune rejection remains similar. The currentlyused Edmonton protocol for islet transplantation is continuallyimproving by using the portal vein and liver for implantation ofisolated islets. Better preservation of isolated islets before and aftertransplantation continues to be a challenge that can be met by newtreatment that protects transplanted beta cells from autoimmune attack.

Given the side effects of insulin therapy and current immunosuppressiveregimens, the search for new therapeutic approaches continues. Therequisite roles of islet-specific autoreactive T and B cells have beenwell established and have been the primary target of current clinicalinvestigations. Building on the role of adaptive immunity, both Tcell-directed immunotherapy with anti-CD3 and the B cell-directed actionof rituximab (anti-CD20) have shown similar efficacy in delaying theprogression of new-onset diabetes. Unfortunately, while clinical benefitto patients in these trials has been recorded (Herold et al. (2005)Diabetes 54: 1763-1769), insulin-secreting capacity continues to declinein treated individuals and these regimens have not restored stabletolerance to islet tissue, perhaps because they do not completely targetthe islet-destructive autoimmune inflammatory process. New therapiesthat protect islets from autoimmune destruction and allow continuinginsulin production are needed.

BRIEF SUMMARY

Described herein are compositions, methods and kits for preserving theviability of insulin-producing beta cells. As shown in the experimentsdescribed herein, a Nuclear Transport Modifier (NTM) in the form of acell-penetrating cSN50 peptide or cSN50.1 peptide affords in vivo isletprotection following a 2-day course of intense treatment in NOD mice,which resulted in a diabetes-free state for one year without apparenttoxicity. cSN50 precipitously reduces the accumulation ofislet-destructive autoreactive B and T lymphocytes while enhancingactivation-induced cell death of T and B lymphocytes derived fromautoimmune diabetes-prone, non-obese diabetic (NOD) mice that developT1D. cSN50 provided attenuation of pro-inflammatory cytokine andchemokine production in immune cells in this model of human T1D. NTMalso provides cytoprotection of beta cells, therefore preservingresidual insulin-producing capacity before and after transplantation.

Unless otherwise defined, all technical terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs.

The terms “patient,” “subject” and “individual” are used interchangeablyherein, and mean an animal (e.g., mammalian (such as human, equine,bovine, ovine, porcine, canine, etc.), reptilian, piscine, etc.) to betreated, diagnosed and/or to obtain a biological sample from.

As used herein, “bind,” “binds,” or “interacts with” means that onemolecule recognizes and adheres to a particular second molecule in asample or organism, but does not substantially recognize or adhere toother structurally unrelated molecules in the sample. Generally, a firstmolecule that “specifically binds” a second molecule has a bindingaffinity greater than about 10⁸ to 10¹² moles/liter for that secondmolecule and involves precise “hand-in-a-glove” docking interactionsthat can be covalent and noncovalent (hydrogen bonding, hydrophobic,ionic, and van der Waals).

As used herein, the term “insulitis” means a lymphocytic invasion andinflammation of the islets of Langerhans in the pancreas.

By the phrase “nuclear transport modifier” and “NTM” is meant a peptidethat is capable of modulating entry of transcription factors into thenucleus. An example of a nuclear transport modifier is a 26-29 aminoacid peptide derived from human nuclear factor kappa B1 nuclearlocalization sequence and from human Fibroblast Growth Factor 4 signalsequence hydrophobic region. This phrase is used interchangeably withthe phrase “nuclear import inhibitor.”

As used herein, “protein” and “polypeptide” are used synonymously tomean any peptide-linked chain of amino acids, regardless of length orpost-translational modification, e.g., glycosylation or phosphorylation.

By the term “gene” is meant a nucleic acid molecule that codes for aparticular protein, or in certain cases, a functional or structural RNAmolecule.

As used herein, a “nucleic acid” or a “nucleic acid molecule” means achain of two or more nucleotides such as RNA (ribonucleic acid) and DNA(deoxyribonucleic acid).

The term “labeled,” with regard to a nucleic acid, protein, probe orantibody, is intended to encompass direct labeling of the nucleic acid,protein, probe or antibody by coupling (i.e., physically or chemicallylinking) a detectable substance (detectable agent) to the nucleic acid,protein, probe or antibody.

As used herein, the terms “therapeutic,” and “therapeutic agent” areused interchangeably, and are meant to encompass any molecule, chemicalentity, composition, drug, cell(s), therapeutic agent, chemotherapeuticagent, or biological agent capable of preventing, ameliorating, ortreating a disease or other medical condition. The term includes smallmolecule compounds, antisense reagents, siRNA reagents, antibodies,enzymes, peptides organic or inorganic molecules, cells, natural orsynthetic compounds and the like.

As used herein, the term “treatment” is defined as the application oradministration of a therapeutic agent to a patient or subject, orapplication or administration of the therapeutic agent to an isolatedtissue or cell line from a patient or subject, who has a disease, asymptom of disease or a predisposition toward a disease, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve or affect the disease, the symptoms of disease, or thepredisposition toward disease.

As used herein, the term “viability” is defined as the ability to live.In the case of insulin-producing beta cells, functional viability ofsuch cells can further include insulin-producing capacity whenappropriately stimulated.

Accordingly, described herein is a method of preserving viability ofinsulin-producing beta cells in pancreatic islets in a mammalian subjectincluding administering a composition including a Nuclear TransportModifier to the mammalian subject, wherein insulin producing capacity ofthe insulin-producing beta cells in the pancreatic islets is preserved.The Nuclear Transport Modifier may be, for example, cSN50 having thesequence set forth in SEQ ID NO:1 or cSN50.1 having the sequence setforth in SEQ ID NO:2. The composition is typically in an amountsufficient to reduce accumulation of autoreactive lymphocytes in thepancreatic islets and pancreatic lymph nodes and enhance cell death ofautoreactive lymphocytes in the pancreatic islets, as well as to enhanceexpression of at least one anti-inflammatory cytokine (e.g., IL-10) inthe pancreatic islets. Administration of the composition decreasesautoimmune inflammation by attenuating expression of at least onestress-responsive transcription factor-regulated gene, and autoimmuneinflammation-induced apoptosis of the insulin-producing beta cells isdecreased or prevented. In the method, sensitivity to activation-inducedcell death in T and B lymphocytes that infiltrate pancreatic islets isgenerally restored. The mammalian subject may be one genetically proneto develop Type 1 diabetes and the composition may be administered tothe mammalian subject before detection of hyperglycemia or afterdetection of hyperglycemia in the subject. Also in the method, thecomposition can be administered to the mammalian subject at one or moreof the following time points: prior to the mammalian subject receiving atransplant of insulin-producing beta cells, concomitant with themammalian subject receiving a transplant of insulin-producing betacells, and subsequent to the mammalian subject receiving a transplant ofinsulin-producing beta cells.

Also described herein is a method of treating diabetes in a mammaliansubject including administering a composition including a NuclearTransport Modifier to the mammalian subject in an amount sufficient forpreserving insulin-producing capacity of beta cells in the mammaliansubject's pancreas and for at least one of: reducing accumulation ofautoreactive lymphocytes in the subject's pancreas; enhancing expressionof anti-inflammatory cytokines in the subject's pancreas; and restoringtolerance to pancreatic islets autoantigens. Typically, administrationof the composition results in remission of the diabetes in the mammaliansubject. The composition may be delivered via any suitable route, e.g.,to the mammalian subject's pancreas. The Nuclear Transport Modifier maybe, for example, cSN50 having the sequence set forth in SEQ ID NO:1 orcSN50.1 having the sequence set forth in SEQ ID NO:2. In one embodiment,the mammalian subject has type 1 diabetes, and the remission occurs inthe presence of insulin therapy. In another embodiment, the mammaliansubject has type 1 diabetes, and the remission occurs in the absence ofinsulin therapy. The composition is in an amount sufficient to enhanceexpression of at least one anti-inflammatory cytokine (e.g., IL-10) inthe pancreatic islets. Administration of the composition decreasesautoimmune inflammation by attenuating expression of at least onestress-responsive transcription factor-regulated gene, and autoimmuneinflammation-induced apoptosis of the insulin-producing beta cells isdecreased or prevented. In the method, sensitivity to activation-inducedcell death in T and B lymphocytes that infiltrate pancreatic islets istypically restored. The composition may be administered to the mammaliansubject at one or more of the following time points: prior to themammalian subject receiving a transplant of insulin-producing betacells, concomitant with the mammalian subject receiving a transplant ofinsulin-producing beta cells, and subsequent to the mammalian subjectreceiving a transplant of insulin-producing beta cells. In anotherembodiment of this method of treating diabetes in a mammalian subject,the composition is in an amount sufficient for preservinginsulin-producing capacity of beta cells in the mammalian subject'spancreas when administered in combination with insulin and for at leastone of: reducing accumulation of autoreactive lymphocytes in thesubject's pancreas; enhancing expression of anti-inflammatory cytokinesin the subject's pancreas; and restoring tolerance to pancreatic isletsautoantigens. In this method, administration of the composition incombination with the insulin results in remission of the diabetes in themammalian subject.

Further described herein is a method of preserving insulin-producingbeta cells including delivering a composition including a NuclearTransport Modifier to a population of insulin-producing beta cells in anamount sufficient to preserve viability of the insulin-producing betacells and the insulin-producing capacity of the insulin-producing betacells if the insulin-producing beta cells are subsequently exposed toproinflammatory stimuli. The Nuclear Transport Modifier may be, forexample, cSN50 having the sequence set forth in SEQ ID NO:1, or cSN50.1having the sequence set forth in SEQ ID NO:2. In some embodiments, thepopulation of insulin-producing beta cells is to be transplanted into amammalian subject in need thereof, and the composition is delivered tothe population of insulin-producing beta cells prior to transplantation.In these embodiments, a pancreas including the population ofinsulin-producing cells may be transplanted into the mammalian subject,and the composition is in an amount sufficient to reduce accumulation ofautoreactive lymphocytes in the pancreatic islets and pancreatic lymphnodes and enhance cell death of autoreactive lymphocytes in thepancreatic islets. Typically, the composition is in an amount sufficientto enhance expression of at least one anti-inflammatory cytokine (e.g.,IL-10) in the pancreatic islets, and to decrease autoimmune inflammationin the mammalian subject by attenuating expression of at least onestress-responsive transcription factor-regulated gene. Generally,autoimmune inflammation-induced apoptosis of the population ofinsulin-producing beta cells is decreased or prevented.

Additionally described herein is a method of treating Type 2 diabetes ina mammalian subject. The method includes administering a compositionincluding a Nuclear Transport Modifier to the mammalian subject havingType 2 diabetes in an amount sufficient for lowering blood glucoselevels and reducing insulin resistance, wherein administration of thecomposition results in remission of the Type 2 diabetes in the mammaliansubject.

Still further described herein is a composition for treating Type 1Diabetes in a subject including a pharmaceutically acceptable carrierand a therapeutically effective amount of Nuclear Transport ModifiercSN50.1 having the sequence set forth in SEQ ID NO:2 sufficient forpreserving viability of insulin-producing beta cells and theinsulin-producing capacity of the cells in the subject.Insulin-producing cells transduced (e.g., transfected) with thecomposition or with Nuclear Transport Modifier cSN50.1 having thesequence set forth in SEQ ID NO:2 are also described herein.

Although compositions, kits, cells, and methods similar or equivalent tothose described herein can be used in the practice or testing of thepresent invention, suitable compositions, kits, cells, and methods aredescribed below. All publications, patent applications, and patentsmentioned herein are incorporated by reference in their entirety. In thecase of conflict, the present specification, including definitions, willcontrol. The particular embodiments discussed below are illustrativeonly and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure. The disclosure may be better understood by reference to oneor more of these drawings in combination with the detailed descriptionof specific embodiments presented herein.

FIGS. 1A-1C show that a Nuclear Transport Modifier suppresses both TCell Receptor- and Toll-Like Receptor-evoked signaling. Splenocytes from10 week old NOD females were isolated and stimulated with anti-CD3/CD28(2 μg/ml), concanavalin (1 μg/ml) (conA), or LPS (5 μg/ml) in thepresence or absence of Nuclear Transport Modifier (cSN50 peptide at 30μM). Supernatants were harvested at 72 h and analyzed. The presence ofIFN-γ as a measure of T cell activation using cytokine bead array wasanalyzed (**p<0.01 vs. cSN50, t-test) (FIG. 1A). Up-regulation of CD80(B7.1) as a measure of B cell responsiveness was assessed by flowcytometry (**p<0.01, Student's t-test) on cells co-expressing CD19 as aB cell marker (FIG. 1B). Pro-inflammatory cytokine production in bonemarrow derived-macrophages cultured in L-conditioned media was examined.Differentiated cells were stimulated with LPS (10 ng/ml) in the presenceor absence of cSN50 (30 μM). cSN50 inhibited production of TNF-α, IL-1α,and IL-1β (*p<0.05, Student's t-test) (FIG. 1C). Data is representativeof three or more experiments.

FIGS. 2A-2F show that in vivo intracellular delivery of a NuclearTransport Modifier to the pancreas attenuates insulitis. To track invivo delivery of NTM, cSN50 was conjugated to fluorescein isothiocyanate(FITC) per the manufacturer's instructions. Animals received one i.p.injection of FITC-cSN50 or an amount of FITC of equivalent relativefluorescence. Animals were euthanized after 2 h and 10 μm frozensections were cut and assessed by confocal microscopy. The cSN50-FITCpeptide is distributed throughout the pancreas. In contrast, FITC alonedid not penetrate the organ and only the autofluorescent tissue borderis seen. Pancreas is shown at 40× magnification (FIG. 2A). cSN50 therapyattenuates ongoing insulitis (FIGS. 2B-2D). 10 week-old NOD femalesreceived one injection of cyclophosphamide (Cy, 0.2 mg/g). 45 h afterthis injection, treatment was begun with cSN50 (35 μg/g every two hours)and continued for 24 h at which time the seven animals in each group(treatment and control) were euthanized and pancreata obtained. Sectionswere stained for T lymphocytes (FIG. 2B) with anti-CD3-PE and for Blymphocytes (FIG. 2C) with anti-B220. The differential interferencecontrast (DIC or Nomarski) image is shown in panel (FIG. 2D). Of 7animals assessed in each group, four NOD mice receiving cSN50 werecompletely insulitis free as compared to persistent insulitis in allcontrol group animals (p<0.05, chi-square). All NOD mice receivinghistologic evaluation in cSN50-treated and control groups (n=7 for each)were assigned insulitis scores based on the severity of inflammation(0=no invading cells, 1=peri-insulits, 2=invasive insulitis, 3=minimalresidual islet tissue). Comparison of control to cSN50-treated animalsshows significantly more unaffected islets in treated mice (*p<0.05) andsignificantly fewer severely affected islets (**p<0.001) (FIG. 2E).Three animals were assessed at 1 year follow-up for insulin productionand insulitis. Sections were stained with a combination of anti-insulinFITC and a combination of anti-CD3 and anti-B220PE (FIG. 2F). Inlong-term survivors (right), insulin staining is detected but insulitiswas not observed. As a staining control, islets from 15 week old controlNODs show significant insulitis.

FIGS. 3A-3C show that autoreactive T cells (BDC2.5 lymphocytes)disappear following in vivo treatment with a Nuclear Transport Modifier.Twenty million CFSE-labeled BDC2.5 splenocytes were transferred topre-diabetic NOD recipients who received cSN50 peptide or saline byosmotic pump. (FIG. 3A). On day 4 post-transfer, pancreatic lymph nodecells were harvested and the proliferation profile of CD4+Thy1.1-cells(FIG. 3B) and absolute number of CFSE+Vβ4+ cells (FIG. 3C) within theCD4 compartment was determined. Treated mice showed significantreduction in the absolute number of recovered Vβ4+ cells (*p<0.05,student's t-test). Figures are representative of three separateexperiments.

FIGS. 4A-4C show that activation-induced cell death (AICD) is enhancedfollowing ex vivo intracellular delivery of a Nuclear Transport Modifierto splenocytes. Splenocytes from 10 week-old NOD females were labeledwith CFSE and stimulated with anti-CD3 (0.05 μg/ml or 2 μg/ml) andanti-CD28 (1 μg/ml) in the presence or absence of cSN50 (30 μM). After65 h of culture, samples were harvested and labeled with annexin V-PE,anti-CD8 PerCP, and anti-CD4 APC. For analysis of AICD, activation wasdefined as the achievement of at least one round of division asdetermined by CFSE-dilution; a representative gating strategy is shownin (FIG. 4A); the undivided peak is highlighted and derived fromunstimulated cells in the same experiment. The CFSE division history,represented graphically as the number of mitoses per 10,000 CD4 or CD8 Tcells, demonstrated no change in proliferation in CD4 or CD8 T cells inthe presence of cSN50 (p=NS) (FIG. 4B). Further analysis revealedincreased annexin-V staining on activated cells, indicative of cellularapoptosis, for cSN50-treated CD4 and CD8 T cells (p<0.005, pairedstudent's t-test) (FIG. 4C). Data is representative of at least 4separate experiments.

FIGS. 5A and 5B show that cSN50 restores activation-induced cell death(apoptosis) in NOD CD4 T cells and enhances B cell apoptosis in responseto LPS. Splenocytes from 10-week-old NOD or C57BL/6 female mice wereisolated and stimulated with anti-CD3/28 (2 μg/ml) or 5 μg/ml LPS in thepresence or absence of cSN50 (30 μM). After 70 h culture, cells wereharvested and apoptosis was detected by annexin V PE staining withlymphocyte co-labeling with either anti-CD4 APC (FIG. 5A) or anti-CD19APC (FIG. 5B). NOD splenocytes when stimulated with anti-CD3/28 showedreduced apoptosis as compared to C57BL/6 splenocytes as previouslypublished (*p<0.01, t-test). Addition of cSN50 normalized apoptosis toC57BL/6 levels (p=NS). Unstimulated lymphocytes showed high levels ofapoptosis, as expected for unactivated lymphocytes in culture; thisprocess was not enhanced by cSN50 (FIG. 5A). Exposure to cSN50significantly enhanced sensitivity to cell death in LPS-stimulated Blymphocytes (p<0.005, paired Student's t-test) (FIG. 5B).

FIGS. 6A and 6B show that expression of immunomodulatory cytokines IL-5and IL-10 is modified during treatment with a Nuclear TransportModifier. Blood samples were obtained daily by saphenous vein bleedingbeginning on the day of Cy challenge. Cytokine levels in plasma weredetermined by cytokine bead array and comparison to a standard curve.cSN50-treated mice demonstrate increased IL-5 (p<0.05 ANOVA; subsequentanalysis of individual days by Student's t-test shows *p<0.05 for days 2and 3) during therapy (FIG. 6A). Splenocytes from 10 week-old NODfemales that had received cyclophosphamide were harvested after 24 hr oftreatment with cSN50 or control non-cell-penetrating peptide (cN50) andrestimulated with LPS to assess their cytokine profile. Supernatantswere harvested at 72 h and analyzed by cytokine bead array for presenceof IL-10. Splenocytes from cSN50 treated animals produced increasedlevels IL-10 (*p<0.05) (FIG. 6B). Data are from three separateexperiments.

FIG. 7 shows that short-term intracellular delivery of a NuclearTransport Modifier in vivo protects NOD mice from autoimmune diabetesfor over one year. Ten week old female NOD mice received one dose of Cy(0.2 mg/g) to synchronize autoimmune diabetes progression. After 45 h,intracellular peptide delivery was initiated with cSN50 (35 μg/g) orwith control and continued every 2 h for 48 h. Blood glucose wasassessed twice weekly. cSN50-treated mice (n=20) were significantlyprotected from diabetes progression as compared to saline-treatedcontrol (n=10, p<0.0001) or the non-cell-penetrating peptidecN50-treated control (n=10, p=0.006). A comparison of cSN50-treated vs.the combined control groups as illustrated in FIG. 7 also demonstratedsignificant protection (p=0.0002, log-rank test).

FIGS. 8A and 8B shows that short-term treatment of NOD mice with aNuclear Transport Modifier shows no effect on measured clinicalbiomarkers of liver and kidney toxicity. Ten week old female NOD micereceived one dose of Cy (0.2 mg/g); after 45 h, intracellular peptidedelivery was initiated with cSN50 (35 μg/g) or with control cN50 andcontinued every 2 h for 48 h. Serum was obtained daily from thesaphenous vein and measurements of ALT (FIG. 8A) and BUN (FIG. 8B) wereperformed in 7 mice in each group. No significant differences weredetermined. Measurements for alkaline phosphatase, creatinine, creatinekinase, and total bilirubin were below the limit of detection in bothgroups.

DETAILED DESCRIPTION

Described herein are compositions, methods, and kits for treatingDiabetes (Type 1 and Type 2) and preserving the viability (e.g.,functional viability) of insulin-producing beta cells. It will beappreciated that for simplicity and clarity of illustration, whereconsidered appropriate, reference numerals may be repeated among thefigures to indicate corresponding or analogous elements. In addition,numerous specific details are set forth in order to provide a thoroughunderstanding of the example embodiments described herein. However, itwill be understood by those of ordinary skill in the art that theexample embodiments described herein may be practiced without thesespecific details. In other instances, methods, procedures and componentshave not been described in detail so as not to obscure the embodimentsdescribed herein.

A novel form of immunotherapy that targets nuclear import as describedherein can arrest inflammation-driven destruction of insulin-producingbeta cells at the site of autoimmune attack within pancreatic isletsduring the progression of T1D. With respect to T1D progression,pro-inflammatory signaling initiated through stimulation of theprincipal receptors of innate immunity—Toll-like receptors (TLRs)—is onemechanism that activates antigen-presenting cells (APCs). In turn, theseeffectors of innate immunity render effector T cells resistant toregulatory T cell (Treg)-mediated suppression. Moreover, autoreactive Bcells that recognize insulin and other autoantigens through B cellreceptor (BCR) can also escape regulatory mechanism and persist inautoimmune diabetes. As a consequence, loss of peripheral toleranceensues. This loss is consistent with reports that naïve T cells in NODmice are resistant to Treg action. Given their escape from bothperipheral and central selection processes, autoreactive T and B cellsgo on to produce critical pro-inflammatory cytokines TNF-α, IL-1β, andIFN-γ that can lead directly to beta cell programmed cell death(apoptosis) (Eizirik D L, and Mandrup-Poulsen T (2001) Diabetologia 44:2115-2133).

Production of these islet-toxic cytokines depends on tightly-regulatedintracellular signal transduction by stress-responsive transcriptionfactors (SRTFs), such as NF-κB, AP-1, NF-AT, STAT-1, and others. NF-κBis the paradigmatic SRTF and has a role in diabetes pathogenesis withcrucial roles played at the levels of both lymphocytes and beta cells.However, other SRTFs, including NF-AT, AP-1, and STAT-1, have also beenimplicated by activating numerous target genes that encode mediators ofinflammation and apoptosis, which underlie destruction of islets andother target tissues. These positive effectors of pro-inflammatoryimmune signaling to the nucleus participate in an auto-stimulatory loop,which amplifies the inflammatory process initiated by microbial andautoimmune triggers. To carry out these potential diabetogenicfunctions, activated SRTFs are ferried to the nucleus of cellsresponding to innate and adaptive immune stimulation. Thus, uncontrollednuclear translocation of SRTFs represents an additional feature of thedysregulated immunity of the murine model of Type 1 Diabetes that maydisrupt peripheral tolerance. The genetic ablation of the common TLRadaptor MyD88 protects NOD mice from autoimmune diabetes (Wen L et al2008 Nature-cited in our PLOS ONE paper as ref#15). A nuclear transportcheckpoint mediated by importins alpha and beta is positioned downstreamof MyD88 thereby serving as a common nexus in the innate and adaptiveimmunity pathways that signal to the nucleus (Hawiger J 2001 ImmunolRes23: 99-109). This culminating step in TLR-evoked innate immunity andBCR- and TCR-evoked adaptive immunity is mediated by nuclear importadaptors known as importins/karyopherins (Hawiger J (2001) Immunol Res23: 99-109).

Targeting nuclear import of stress-responsive transcription factorsevoked by agonist-stimulated innate and adaptive immunity receptorsprotects islets from autoimmune destruction. The new mode of T1D controldisclosed herein allows simultaneous inhibition of TLR-evoked innateimmunity and T cell receptor (TCR)-initiated adaptive immunity. Acomposition and method to target importins/karyopherins by inhibitingnuclear transport offers a new level of control toward dysregulatedinnate and adaptive immune signaling in T1D. Since both immune responsesdepend on intracellular signal transduction by SRTFs, the nuclear importmechanism was targeted with a cell-penetrating Nuclear TransportModifier. Short-term intracellular delivery of this inhibitor affordedlong-term protection of the islets from inflammation-driven apoptosis.This long-lasting (one year) islet-protecting effect, which arrestsdiabetes progression without the need for insulin therapy, appears toinvolve the precipitous reduction of autoreactive lymphocytes throughenhancement of AICD of T and B lymphocytes. Moreover, this salutaryeffect of short-term nuclear import targeting is associated withreprogramming of the pro-inflammatory and anti-inflammatory cytokineprofile of immune cells isolated from non-obese diabetic (NOD) mice.

The ultimately fatal outcome of autoimmune diabetes in the widely usedand clinically relevant murine NOD model of human T1D depends onprogressive and relentless destruction of insulin-producing beta cellsin pancreatic islets and is inevitable unless insulin-replacementtherapy is instituted. Islets are protected from autoimmune attack byintracellular delivery of a Nuclear Transport Modifier peptide such ascSN50 and cSN50.1. These peptides effectively protected islets fromimmune destruction in the experiments described herein. This protectionis vested in significant reduction of islet-reactive T cells,restoration of the sensitivity of T and B cells to activation-inducedcell death, suppression of islet-toxic pro-inflammatory cytokineproduction in primary T and B cells and macrophages isolated from NODmice, and preservation of a key anti-inflammatory cytokine, IL-10. Thus,a Nuclear Transport Modifier extinguished autoimmune inflammation-drivenislet loss and prevented further progression of diabetes therebyobviating the need for insulin replacement therapy during a one-yearobservation period. Because intracellular delivery of a NuclearTransport Modifier peptide such as cSN50 and cSN50.1 can result inlowering of blood glucose levels and can reduce insulin resistance, thecompositions, methods and cells described herein can also be used fortreating Type 2 diabetes (T2D).

Nuclear Transport Modifiers include but are not limited to cSN50,cSN50.1, and SN50. Any peptide that is capable of modulating entry intothe nucleus of stress-responsive transcription factors (SRTFs) may be aNuclear Transport Modifier. cSN50 is a cyclic peptide combining thehydrophobic region of the Kaposi fibroblast growth factor signalsequence with the nuclear localization signal (NLS) of the p50-NFκB1 andinserting a cysteine on each side of the NLS to form an intrachaindisulfide bond. The amino acid sequence of cSN50 isAAVALLPAVLLALLAPCYVQRKRQKLMPC (SEQ ID NO:1). Methods of making and usingcSN50 are described, for example, in U.S. Pat. Nos. 7,553,929 and6,495,518. In another embodiment, cSN50.1 may be administered to protectislets from immune destruction and preserve viability ofinsulin-producing beta cells. cSN50.1 is a cyclic peptide having thesequence of cSN50 with the exception that the tyrosine at position 18 ofcSN50, adjacent to the first cysteine, has been removed. The amino acidsequence of cSN50.1 is AAVALLPAVLLALLAPCVQRKRQKLMPC (SEQ ID NO:2).cSN50.1 was designed to increase the solubility of the cSN50 peptide;i.e., a tyrosine was removed from the sequence of cSN50 to increasesolubility. Additional examples of NTMs include synthesized peptides inwhich cargo is incorporated as two, rather than one, modules or cargosderived from intracellular proteins other than NFκB1.

In the experiments described herein, none of the cSN50 peptide-treatedanimals developed hyperglycemia during the first phase of diabetes onsetoccurring between days 10-30 after receiving a bolus of cyclophosphamide(Cy), which synchronized the autoimmune diabetes process in NOD mice. Inaddition to this early protective effect, cSN50 treatment also affordedsignificant long-term islet protection. While another half ofCy-synchronized control animals progressed to diabetes between days 50and 100, only two of twenty cSN50-treated animals developed diabetes, afinding suggesting that cSN50 treatment resulted in long-term isletprotection in NOD mice that are genetically-prone to T1D. This favorableoutcome is supported by the demonstration of in vivo elimination ofislet-infiltrating and islet-reactive lymphocytes (FIG. 3), most likelythrough enhanced AICD, which was demonstrated ex vivo in cSN50peptide-treated T and B cells derived from diabetes-prone NOD mice (FIG.4). At higher levels of stimulation (e.g., higher concentrations of themitogenic stimulus, anti-CD3), the sensitivity to AICD is furtherincreased by the Nuclear Transport Modifier peptide. Thus, even chronicactivation of islet-infiltrating T and B cells in autoimmune diabetesthat renders NOD mice-derived T and B lymphocytes resistant to AICD maybe counteracted by the AICD-enhancing effect of the Nuclear TransportModifier. This action of NTM may favor rapid elimination of autoreactiveand islet-destructive T and B cell clones in NTM-treated NOD mice. Theaction of cSN50 in a relevant preclinical T1D model adds autoimmuneinflammation to the list of conditions in which a Nuclear TransportModifier has displayed therapeutic utility. Nuclear Transport Modifierdelivery and its anti-inflammatory and cytoprotective action areeffective in acute inflammation models, including lethal challenge withsuperantigen, staphylococcal enterotoxin B (SEB), and lipopolysaccharide(LPS), which trigger acute inflammatory lung and liver injury (Liu etal. (2009) Mol Ther 17: 796-802; Liu et al. (2004) J Biol Chem 279:19239-19246). Moreover, Nuclear Transport Modifiers inhibit nuclearentry of stress-responsive transcription factors, NF-κB, NF-AT, AP-1,and STAT-1 in human T lymphocytes (Hawiger J (1999) Current Opinion inChem. Biology 3:89-94).

An important aspect of the cSN50 Nuclear Transport Modifier is itsability to reach the pancreas (FIG. 2A) and cells comprising pancreaticlymph nodes, as well as other lymphoid and non-lymphoid organs. Themechanism of intracellular delivery of this peptide has been elucidatedand an endocytosis-independent process of crossing the plasma membranemediated by the membrane-translocating motif (MTM), which is based onthe signal sequence hydrophobic region (SSHR) derived from Kaposi FGF,has been documented (Veach et al. (2004) J Biol Chem 279: 11425-11431).The amphipathic helix-based structure of SSHR facilitates its insertiondirectly into the plasma membrane and the tilted transmembraneorientation permits the translocation of the Nuclear Transport Modifierthrough the phospholipid bilayer of the plasma membrane directly to theinterior of the cell without perturbing membrane integrity. Thismechanism explains the efficient delivery of SSHR-guided cargo acrossthe plasma membrane of multiple cell types involved in autoimmuneinflammation.

This presents a new avenue for altering the course of diabetesprogression as there has been limited success in obviating the need forparenteral insulin-replacement therapy of T1D to date. A broadrepertoire of SRTFs-regulated genes that encode mediators of isletinflammation and beta cells apoptosis is attenuated. Contributing to theshort-circuiting of this pro-inflammatory signaling cascade, nuclearimport modulation reversed resistance of autoreactive T cells to AICD.Indeed, as islet-reactive lymphocytes are likely to be maximallystimulated during disease progression, in the experiments describedherein, cSN50 enhanced their deletion as compared to those lymphocyteswithout islet-reactive specificities (compare FIGS. 2, 3, 5). Thus,cSN50 treatment seems to restore peripheral T and B cell tolerance,which critically depends on the appropriate regulation of lymphocyteAICD in addition to recovery of physiologically immunosuppressiveregulatory T cells (Tregs). Tregs are the mainstays of peripheraltolerance. It is surmised that Nuclear Transport Modifier may counteractinhibition of Treg by inflammatory mediators generated in autoimmunedisorders such as T1D. This NTM effect is supported by enhancedproduction of anti-inflammatory cytokine IL-10 linked to Tregsactivation (see below).

In addition to enhancing autoreactive lymphocyte elimination, theNuclear Transport Modifier may also modulate the cytokine milieuestablished by immune cells in their target organs. cSN50 inhibitspro-inflammatory cytokine expression in ex vivo analyzed NOD splenocyteswhile preserving and even enhancing the anti-inflammatory cytokineIL-10. An increase in IL-5 in the plasma of treated mice during thefirst day of cSN50 therapy was found. Increased levels of IL-4 or IL-13were not found and thus it is unclear whether this increased IL-5 isindicative of a shift towards a Th2 phenotype. While Th2 shifts haveoccasionally been associated with diabetes protection, it is not clearthat this shift is a part of the true protective mechanism in manycases. The pattern of increased IL-10 and IL-5 was also seen in humansubjects in the original trial of anti-CD3 (Herold et al. (2002) N EnglJ Med 346: 1692-1698). Cumulatively, these findings suggest that IL-10and IL-5 may play an important role in modulating the course of Type 1diabetes in tolerized individuals.

Reduction or complete elimination of islet-destructive autoreactive Tand B cells may be useful in treating T1D and potentially otherautoimmune diseases. Other autoimmune disorders include Systemic LupusErythematosus, Multiple Sclerosis, Rheumatoid Arthritis, Crohn Diseases,and Ulcerative Collitis. Under conditions of metabolic, inflammatory,and oxidant stress, beta cell mass may be further controlled byimportant interactions between bone marrow-derived cells and islet betacells. Transcriptional modulation of these interactions via targeting ofthe nuclear import machinery represents an important new opportunity fortherapeutic development. Even though cSN50 can modulate nucleartransport of several stress-responsive transcription factors thatutilize importin alpha 1/karyopherin alpha 5 (as well as otherimportins/karyopherins) for nuclear trafficking, intracellular deliveryof the Nuclear Transport Modifier is rapid and restricted to therelatively short intracellular persistence of the injected cargo(Hawiger J., (1999) Current Opinion in Chem. Biology 3:89-94). Despitehigh intensity dosing of cSN50 in the protocols disclosed herein,short-term or long-term adverse effects were not observed. This suggeststhat this therapy would be tolerated during longer-term application, ifnecessary, to prevent or reverse spontaneous disease. In preliminarystudies, spontaneous disease was reversed in 40% of the NOD mice duringa continuing NTM treatment for 3 weeks. In other disease models,continued cSN50 peptide delivery has been continued for up to 8 weekswithout evidence of toxicity including normal animal health andsurvival, normal weight gain, normal liver enzymes, normal blood cellcounts including leukocyte and lymphocyte subsets, and absence ofapparent infectious illness.

The data presented herein show that short-term intensive targeting ofthe nuclear import shuttle for SRTFs can protect islets from relentlessautoimmune attack and induce long-term remission of T1D in NOD mice withestablished insulitis. Chronic inflammatory destruction of beta cellsmay be reversed without overt signs of toxicity by the intracellularimmunotherapy for T1D disclosed herein. Isolated insulin-producing cellsmay be treated with a Nuclear Transport Modifier or a compositionincluding a Nuclear Transport Modifier to preserve their viability andinsulin-producing capacity. Such treated cells can be used fortransplantation into a subject in need thereof (e.g., a subjectsuffering from diabetes). The Nuclear Transport Modifier disarmsislet-destroying immune cells and prevents apoptosis of beta cellsinduced by proinflammatory, autoimmune, and oxidant stress.Intracellular delivery of a Nuclear Transport Modifier to NOD protectstheir insulin-producing beta cells from immune injury and preservesinsulin producing capacity. In cultured beta cell lines, activation ofcell death executioner caspase 3 following a challenge withproinflammatory and proapoptotic stimuli is attenuated by NTM treatment.Disclosed herein is a new method of preserving residualinsulin-producing capacity in Type 1 and Type 2 diabetes, preservinghuman islets before and after transplantation, and treating diabetes.

Biological and Chemical Methods

Methods involving conventional molecular biology techniques aredescribed herein. Such techniques are generally known in the art and aredescribed in detail in methodology treatises (Sambrook et al. ed.,(2001) Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Ausubel et al.ed., (1992) (with periodic updates) Current Protocols in MolecularBiology, ed., Greene Publishing and Wiley-Interscience, New York).

Compositions for Treating Diabetes in a Subject

Compositions, e.g., pharmaceutical compositions, described herein fortreating Type 1 Diabetes in a subject (e.g., a human subject) include atherapeutically effective amount of a Nuclear Transport Modifier (suchas cSN50 or cSN50.1) sufficient for preserving viability ofinsulin-producing beta cells and the insulin-producing capacity of thecells and a pharmaceutically acceptable carrier. Similarly, compositionsdescribed herein for treating Type 2 Diabetes in a subject (e.g., ahuman subject) include a therapeutically effective amount of a NuclearTransport Modifier (such as cSN50 or cSN50.1) sufficient for loweringblood glucose levels and reducing insulin resistance, and apharmaceutically acceptable carrier. Such compositions may also includea second therapeutic, e.g., insulin.

Method of Preserving Viability of Insulin-Producing Beta Cells inPancreatic Islets in a Mammalian Subject

A typical method of preserving viability of insulin-producing beta cellsin pancreatic islets in a mammalian subject includes administering acomposition including a Nuclear Transport Modifier to the mammaliansubject. In this method, the insulin producing capacity of theinsulin-producing beta cells in the pancreatic islets is preserved. Anysuitable Nuclear Transport Modifier can be used, e.g., cSN50 having thesequence set forth in SEQ ID NO:1, cSN50.1 having the sequence set forthin SEQ ID NO:2, etc. The composition is typically in an amountsufficient to result in one or more of the following: a reduction ofaccumulation of autoreactive lymphocytes in the pancreatic islets andpancreatic lymph nodes; enhancement of cell death of autoreactivelymphocytes in the pancreatic islets; enhancement of expression of atleast one anti-inflammatory cytokine (e.g., IL-10) in the pancreaticislets; a decrease in autoimmune inflammation by attenuating expressionof at least one SRTF-regulated gene; reduction or prevention ofautoimmune inflammation-induced apoptosis of the insulin-producing betacells; and restoration of sensitivity to activation-induced cell deathin T and B lymphocytes that infiltrate pancreatic islets. In the method,the composition can be administered to the mammalian subject at one ormore time points, e.g., prior to diagnosis of T1D in a genetically pronesubject (e.g., a subject whose family has a relatively high incidence ofT1D), after diagnosis of T1D and initiation of insulin therapy, prior tothe subject receiving a transplant of insulin-producing beta cells,concomitant with the subject receiving a transplant of insulin-producingbeta cells, and subsequent to the subject receiving a transplant ofinsulin-producing beta cells.

Method of Treating Type 1 Diabetes in a Subject

The therapeutic methods of the invention in general includeadministration of a therapeutically effective amount of a composition asdescribed herein to a subject (e.g., animal) in need thereof, includinga mammal, particularly a human. Such treatment will be suitablyadministered to subjects, particularly humans, suffering from, orhaving, diabetes or signs thereof. The composition may also be added tobeta cells to protect their viability before and after transplantation.The compositions herein may also be used in the treatment of any otherdisorders in which inflammation-driven destruction of beta cells may beimplicated.

In a typical method of treating Type 1 diabetes in a mammalian subject,the method includes administering a composition including a NuclearTransport Modifier (e.g., cSN50, cSN50.1) to the mammalian subject in anamount sufficient for preserving insulin-producing capacity of betacells in the mammalian subject's pancreas and for at least one of:reducing accumulation of autoreactive lymphocytes in the subject'spancreas; enhancing expression of anti-inflammatory cytokines in thesubject's pancreas; and restoring tolerance to pancreatic isletsautoantigens. Administration of the composition results in remission ofthe diabetes in the mammalian subject. In some embodiments, thecomposition is delivered to the mammalian subject's pancreas. In oneembodiment, the mammalian subject has Type 1 diabetes, and the remissionoccurs in the absence of insulin therapy. In another embodiment, themammalian subject has Type 2 diabetes, and receives insulin therapy incombination with a composition as described herein. In this embodiment,the remission occurs in the presence of insulin therapy. In the methodsdescribed herein, a composition as described herein may also includeinsulin, and thus the Nuclear Transport Modifier and insulin areadministered to the subject as one composition. Alternatively, insulinmay be administered to the subject separately from the compositionincluding a Nuclear Transport Modifier, either at the same time(concomitantly) or at different time points. Insulin and compositionsincluding insulin can be administered by any suitable route. Methods ofdelivering insulin are well known in the art.

For treating diabetes, the compositions described herein can beadministered at any appropriate time point. For example, a compositionmay be administered before, during and after a transplantation (e.g.,islet transplantation, pancreas transplantation). In one embodiment, acomposition is administered before and after transplantation (e.g., of apancreas or islets), thereby suppressing an immune attack on thetransplanted organ or cells and prolonging their lifespan. As additionalexamples, a composition may be administered to a subject (e.g., a humanpatient) known to be genetically prone to T1D and before hyperglycemia(diabetes) is apparent, after hyperglycemia is first detected, and whenhyperglycemia is well established (this means that all pancreatic isletsare destroyed). At the time when hyperglycemia is well established, apatient may be a candidate for pancreas or islet transplantation andadministration of the compositions described herein and employment ofthe methods described herein should “prime” the patient for pancreas orislet transplantation. Thereafter, such treatment should continue toprotect a transplanted pancreas or islets from beta cell-directedinjury. In some embodiments, use of the compositions, cells and methodsdescribed herein may promote or enable transplantation by reducingautoreactive B and T cells in a patient before receipt of the organ orislets (to be transplanted), i.e., suppressing an immune attack on thetransplanted organ or islets, thereby preserving transplanted cell(e.g., islet cells) viability by shielding them from autoreactive B andT lymphocytes.

In some embodiments, a subject's response to the compositions describedherein (and optionally to insulin therapy as well) is measured. In oneembodiment, the invention provides a method of monitoring treatmentprogress. The method includes the step of determining a level ofexpression of diagnostic markers such as blood glucose, C-peptide,anti-insulin antibody, IFN-γ, TNF-α, IL-1α, IL-1β, and IL-10 (e.g., anytarget delineated herein modulated by a composition or agent describedherein, a protein or indicator thereof, etc., or diagnostic measurement(e.g., screen, assay)) in a subject suffering from or susceptible to adisorder or symptoms thereof associated with Type 1 Diabetes in whichthe subject has been administered a therapeutic amount of a compositionas described herein for treating the disease or symptoms thereof. Thelevel of marker determined in the method can be compared to known levelsof marker in either healthy normal controls or in other afflictedpatients to establish the subject's disease status. In preferredembodiments, a second level of marker (e.g., C-peptide, anti-insulinantibody, IFN-γ, TNF-α, IL-1α, and IL-1β, and IL-10) in the subject isdetermined at a time point later than the determination of the firstlevel, and the two levels are compared to monitor the course of diseaseor the efficacy of the therapy. In certain preferred embodiments, apre-treatment level of marker in the subject is determined prior tobeginning treatment according to the methods described herein; thispre-treatment level of marker can then be compared to the level ofmarker in the subject after the treatment commences, to determine theefficacy of the treatment.

Also described herein are diagnostic and theranostic methods useful todetermine whether the subject or beta cells are susceptible to thetreatment methods of the invention. The term “theranostics” generallyrefers to therapy-specific diagnostics, which is the use of diagnostictesting to diagnose the disease, choose the correct treatment regime forthat disease, and monitor the patient response to therapy. Theranostictests can be used to predict and assess drug response in individualpatients, and are designed to improve drug efficacy by selectingpatients for treatments that are particularly likely to benefit from thetreatments. Theranostic tests are also designed to improve drug safetyby identifying patients that may suffer adverse side effects from thetreatment.

Method of Treating Type 2 Diabetes in a Subject

In some embodiments, the methods described herein are used to treatsubjects susceptible to or suffering from Type 2 diabetes. In suchembodiments, not only are beta cells protected from metabolic insultse.g. glucotoxicity, but insulin resistance may be reduced. Intracellulardelivery of a Nuclear Transport Modifier peptide such as cSN50 andcSN50.1 can result in lowering of blood glucose levels and may reduceinsulin resistance. In a typical method of treating Type 2 diabetes in amammalian subject, the method includes administering a compositionincluding a Nuclear Transport Modifier (e.g., cSN50, cSN50.1) to themammalian subject in an amount sufficient for lowering blood glucoselevels and reducing insulin resistance, and a pharmaceuticallyacceptable carrier. In a subject suffering from Type 2 diabetes,administration of the composition results in remission of the diabetesin the mammalian subject.

Kits

Described herein are kits for treating Type 1 diabetes in a subject andprotecting beta cells (in vitro, in vivo, ex vivo, in pancreaticislets). Such a kit may be particularly useful for preserving beta cellviability and function of such cells before and after transplantation.In one embodiment, a kit includes: a composition including apharmaceutically acceptable carrier, a Nuclear Transport Modifier suchas cSN50 or cSN50.1, packaging, and instructions for use. In thecomposition, the amount of Nuclear Transport Modifier such as cSN50 orcSN50.1 is sufficient for treating Type 1 Diabetes or preserving theviability and function of beta cells in pancreatic islets. In anotherembodiment, a kit includes beta cells which have been treated with acomposition as described herein. Also described herein are kits treatingType 2 diabetes. In such an embodiment, a kit can include apharmaceutically acceptable carrier, packaging, instructions for use,and a Nuclear Transport Modifier (e.g., cSN50, cSN50.1) in an amountsufficient for lowering blood glucose levels and reducing insulinresistance. Optionally, kits may also contain one or more of thefollowing: containers which include positive controls, containers whichinclude negative controls, photographs or images of representativeexamples of positive results and photographs or images of representativeexamples of negative results.

Administration of Compositions and Cells

The administration of a composition (e.g., a pharmaceutical composition)including a Nuclear Transport Modifier such as cSN50 or cSN50.1 in anamount effective for, for example, treating Type 1 Diabetes in a subjectand protecting beta cells before and after transplantation, may be byany suitable means that results in a concentration of the therapeuticthat is effective. cSN50 or cSN50.1 may be contained in any appropriateamount in any suitable carrier substance, and are generally present inan amount of 1-95% by weight of the total weight of the composition. Thecomposition may be provided in a dosage form that is suitable for localor systemic administration (e.g., parenteral, subcutaneously,intravenously, intramuscularly, or intraperitoneally, intrahepatically).The pharmaceutical compositions may be formulated according toconventional pharmaceutical practice (see, e.g., Gennaro, A. R. ed.(2000) Remington: The Science and Practice of Pharmacy (20th ed.),Lippincott Williams & Wilkins, Baltimore, Md.; Swarbrick, J. and Boylan,J. C., eds. (1988-1999) Encyclopedia of Pharmaceutical Technology,Marcel Dekker, New York).

Compositions as described herein may be administered parenterally byinjection, infusion or implantation (subcutaneous, intravenous,intramuscular, intraperitoneal, intrahepatic, or the like) in dosageforms, formulations, or via suitable delivery devices or implantscontaining conventional, non-toxic pharmaceutically acceptable carriersand adjuvants. The formulation and preparation of such compositions arewell known to those skilled in the art of pharmaceutical formulation.Formulations can be found in, for example, Gennaro, A. R. supra.

Compositions for parenteral use may be provided in unit dosage forms(e.g., in single-dose ampoules), or in vials containing several dosesand in which a suitable preservative may be added (see below). Thecomposition may be in the form of a solution, a suspension, an emulsion,an infusion device, or a delivery device for implantation, or it may bepresented as a dry powder to be reconstituted with water or anothersuitable vehicle before use. Apart from the active agent that treatsType 1 Diabetes and protects beta cells (e.g., before and aftertransplantation), and that treats Type 2 Diabetes and lowers bloodglucose levels, the composition may include suitable parenterallyacceptable carriers and/or excipients. The active therapeutic agent(s)may be incorporated into microspheres, microcapsules, nanoparticles,liposomes, or the like for controlled release. Furthermore, thecomposition may include suspending, solubilizing, stabilizing,pH-adjusting agents, tonicity adjusting agents, and/or dispersingagents.

As indicated above, the pharmaceutical compositions described herein maybe in a form suitable for sterile injection. To prepare such acomposition, the suitable active therapeutic(s) are dissolved orsuspended in a parenterally acceptable liquid vehicle. Among acceptablevehicles and solvents that may be employed are water, water adjusted toa suitable pH by addition of an appropriate amount of hydrochloric acid,sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer'ssolution, and isotonic sodium chloride solution and dextrose solution.The aqueous formulation may also contain one or more preservatives(e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). In cases where oneof the compounds is only sparingly or slightly soluble in water, adissolution enhancing or solubilizing agent can be added, or the solventmay include 10-60% w/w of propylene glycol or the like.

Materials for use in the preparation of microspheres and/ormicrocapsules are, e.g., biodegradable/bioerodible polymers such aspolygalactin, poly-(isobutyl cyanoacrylate),poly(2-hydroxyethyl-L-glutamine), and poly(lactic acid). Biocompatiblecarriers that may be used when formulating a controlled releaseparenteral formulation are carbohydrates (e.g., dextrans), proteins(e.g., albumin), lipoproteins, or antibodies. Materials for use inimplants can be non-biodegradable (e.g., polydimethyl siloxane) orbiodegradable (e.g., poly(caprolactone), poly(lactic acid),poly(glycolic acid) or poly(ortho esters) or combinations thereof).

Formulations for oral use include tablets containing the activeingredient(s) (e.g., cSN50 or cSN50.1) in a mixture with non-toxicpharmaceutically acceptable excipients. Such formulations are known tothe skilled artisan. Excipients may be, for example, inert diluents orfillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystallinecellulose, starches including potato starch, calcium carbonate, sodiumchloride, lactose, calcium phosphate, calcium sulfate, or sodiumphosphate); granulating and disintegrating agents (e.g., cellulosederivatives including microcrystalline cellulose, starches includingpotato starch, croscarmellose sodium, alginates, or alginic acid);binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid,sodium alginate, gelatin, starch, pregelatinized starch,microcrystalline cellulose, magnesium aluminum silicate,carboxymethylcellulose sodium, methylcellulose, hydroxypropylmethylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethyleneglycol); and lubricating agents, glidants, and antiadhesives (e.g.,magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenatedvegetable oils, or talc). Other pharmaceutically acceptable excipientscan be colorants, flavoring agents, plasticizers, humectants, bufferingagents, and the like.

The tablets may be uncoated or they may be coated by known techniques,optionally to delay disintegration and absorption in thegastrointestinal tract and thereby providing a sustained action over alonger period. The coating may be adapted to release the active drug ina predetermined pattern (e.g., in order to achieve a controlled releaseformulation) or it may be adapted not to release the active drug untilafter passage of the stomach (enteric coating). The coating may be asugar coating, a film coating (e.g., based on hydroxypropylmethylcellulose, methylcellulose, methyl hydroxyethylcellulose,hydroxypropylcellulose, carboxymethylcellulose, acrylate copolymers,polyethylene glycols and/or polyvinylpyrrolidone), or an enteric coating(e.g., based on methacrylic acid copolymer, cellulose acetate phthalate,hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcelluloseacetate succinate, polyvinyl acetate phthalate, shellac, and/orethylcellulose). Furthermore, a time delay material, such as, e.g.,glyceryl monostearate or glyceryl distearate may be employed.

The solid tablet compositions may include a coating adapted to protectthe composition from unwanted chemical changes, (e.g., chemicaldegradation prior to the release of the active therapeutic substance).The coating may be applied on the solid dosage form in a similar manneras that described in Swarbrick, J. and Boylan, J. C., supra. At leasttwo therapeutics (e.g., a composition including cSN50 or cSN50.1, aswell as a second anti-Type 1 Diabetes or islet transplantationtherapeutic or anti-Type 2 Diabetes therapeutic) may be mixed togetherin the tablet, or may be partitioned. In one example, the first activetherapeutic is contained on the inside of the tablet, and the secondactive therapeutic is on the outside, such that a substantial portion ofthe second active therapeutic is released prior to the release of thefirst active therapeutic.

Formulations for oral use may also be presented as chewable tablets, oras hard gelatin capsules wherein the active ingredient is mixed with aninert solid diluent (e.g., potato starch, lactose, microcrystallinecellulose, calcium carbonate, calcium phosphate or kaolin), or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example, peanut oil, liquid paraffin, or olive oil.Powders and granulates may be prepared using the ingredients mentionedabove under tablets and capsules in a conventional manner using, e.g., amixer, a fluid bed apparatus or a spray drying equipment. Compositionsas described herein can also be formulated for inhalation and topicalapplications. Optionally, an anti-diabetes or transplantationtherapeutic may be administered in combination with any other standardanti-diabetes or transplantation therapy; such methods are known to theskilled artisan (see, e.g., Gennaro, supra). Combinations are expectedto be advantageously synergistic. Therapeutic combinations that preserveviability of insulin-producing beta cells are identified as useful inthe compositions and methods described herein.

In ex vivo methods of preserving insulin-producing beta cells in theislets prepared for transplantion and treating diabetes, islets treatedwith a composition as described herein can be delivered (e.g.,transplanted) to a subject via any suitable means. One or more of thedelivery means described herein for administering compositions can beused for delivery of treated islets to a subject. In a typicalembodiment of islet transplantation, the currently used Edmontonprotocol (see, e.g., N Engl J Med 2006; 355:1318-1330) involving use ofthe portal vein and liver for implantation of isolated islets may beused. However, any suitable method of islet transplantation may be used.Islets may be obtained from any suitable source. For example, two tofour whole pancreases from human donors may be used for a single isletcell transplant implanted in the recipient's liver.

The therapeutic methods described herein in general includeadministration of a therapeutically effective amount of the compositionsand/or cells (e.g., islets, pancreas) described herein to a subject(e.g., animal) in need thereof, including a mammal, particularly ahuman. Such treatment will be suitably administered to subjects,particularly humans, suffering from, having, susceptible to, or at riskfor Type 1 Diabetes or beta cell destruction or at risk for Type 2Diabetes and insulin resistance and elevated glucose levels.Determination of those subjects “at risk” can be made by any objectiveor subjective determination by a diagnostic test or opinion of a subjector health care provider. The methods, cells, and compositions herein maybe also used in the treatment of any disorders in whichinflammation-driven destruction of cells may be implicated.

Effective Doses

The compositions (e.g., pharmaceutical compositions) and cells (e.g.,islets, pancreas) described herein are preferably administered to ananimal (e.g., mammalian (such as human), reptilian, piscine, etc.) in aneffective amount, that is, an amount capable of producing a desirableresult in a treated animal (e.g., preserving viability ofinsulin-producing beta cells, treating diabetes, lowering blood glucoselevels, reducing insulin resistance). Such a therapeutically effectiveamount can be determined according to standard methods. Toxicity andtherapeutic efficacy of the compositions utilized in methods of theinvention can be determined by standard pharmaceutical procedures. As iswell known in the medical and veterinary arts, dosage for any onesubject depends on many factors, including the subject's size, bodysurface area, age, the particular composition to be administered, timeand route of administration, general health, and other drugs beingadministered concurrently.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the present disclosure. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventors to function well andthus can be considered to constitute preferred modes for its practice.However, those of skill in the art should, in light of the presentdisclosure, appreciate that many changes can be made in the specificembodiments which are disclosed and still obtain a like or similarresult without departing from the spirit or scope of the disclosure. Thefollowing Examples are offered by way of illustration and not by way oflimitation.

Example 1 Mice and Diabetes Monitoring

NOD/LtJ, NOD-BDC2.5 and C57BL/6 mice were purchased from the JacksonLaboratories (Bar Harbor, Me.) at 6-8 weeks of age. All mice were housedand maintained according to the guidelines for use and care oflaboratory animals as set forth by Vanderbilt University and regulatedvia the Vanderbilt IACUC. All NOD mice were monitored twice weekly forthe development of diabetes by blood glucose measurement with FreeStyle®FastTake test strips (Abbott Laboratories, Abbott Park, Ill.). Twoconsecutive glucose measurements>220 mg/dl constituted a diagnosis ofdiabetes. A colony of NOD mice kept by a collaborating laboratory in thesame animal suite has a spontaneous diabetes incidence of 80-90% infemales by 30 weeks of age indicating an animal environment that isconducive for diabetes development.

Isolation of Lymphoid Cells and Preparation of Bone Marrow DerivedMacrophages

Splenocytes and lymph node cells were prepared by dispersion of theorgan and passage through a 70-μm cell strainer followed by red celllysis and resuspension in the media of choice for the given experiment.For preparation of primary macrophages, bone marrow from pre-diabetic,8-12 week old female NOD mice was prepared by flushing mouse femurs andtibias with ice-cold DMEM supplemented with L-glutamine. Bone marrowcells were pooled, passed through a 25 5/8-gauge needle, and filteredthrough a 70-μm cell strainer. Pooled cells (1×10⁶ cells/ml) weresuspended in DMEM supplemented with 10% FBS, 10 mM HEPES, penicillin(100 U/ml), streptomycin (100 μg/ml), and 20% L929 conditioned mediumfollowed by plating on 150-mm bacterial Petri dishes. Cells wereincubated at 37° C. in 5% CO₂ in humid air. Every 3 days, non-adherentcells were removed, cells were washed, and culture medium was replaced.Cells were used in experiments after 10 days of culture for up to 2weeks after maturation. When analyzed by flow cytometry, 95% of theadherent cells were MAC3+, CD3−, and B220−. The viability of BMDMswas >80% before use in all experiments.

Ex Vivo Stimulation of NOD Lymphocytes

Cells were plated in 24-well plates at a density of 1×10⁶ total cells/mlin DMEM containing 10% HI-FCS, penicillin (100 U/ml), streptomycin (100μg/ml), 2-mercaptoethanol (55 μM), and varying amounts of the identifiedstimulus including anti-CD3 (0-2 μg/ml) with 1 μg/ml anti-CD28, LPS (5μg/ml), or concanavalin A (1 μg/ml) (conA). All cells were incubated for65-70 h at 37° C. in 5% CO₂.

Synthesis, purification, and labeling of a cell-penetrating peptideinhibitor of nuclear import (cSN50) and its non-cell-penetrating control(cN50)

Cell-penetrating peptide (cSN50, MW=3149; cSN50.1, MW=2986), andnon-cell-penetrating peptide (cN50, MW=1651), were synthesized,purified, filter-sterilized, and analyzed as described elsewhere(Torgerson et al. (1998) J Immunol 161: 6084-6092; Liu et al. (2000) JBiol Chem 275: 16774-16778). To monitor the intracellular delivery ofpeptides to pancreas, cSN50 peptide was coupled with fluoresceinisothiocyanate (FITC, Pierce) according to the manufacturer's protocol.

In Vivo Intracellular Peptide Delivery to the Pancreas

For in vivo detection of fluorescein-labeled peptides in the pancreas,NOD mice were sacrificed at 2 h after intraperitoneal (i.p.) injectionof 0.7 mg of FITC-labeled cSN50 peptide/mouse. The pancreata were washedwith saline and prepared for cryosections (10 μm thickness). Controlsolution of FITC alone with equivalent fluorescence units was injectedseparately to track distribution to the pancreas.

Synchronization of T1D Progression with Cyclophosphamide

At 10-11 weeks of age, female NOD mice received an intraperitoneal bolusinjection of 200 mg/kg cyclophosphamide (Sigma). Cyclophosphamide wasprepared by reconstitution of powder in sterile saline for injection.

Treatment with Nuclear Transport Modifier and Control

45 h after diabetes synchronization with cyclophosphamide, treatmentwith cSN50 or control was initiated. Mice receiving cSN50 were given 35μg/g of cSN50 i.p. every two hours for the next 48 h. Control micereceived either saline or non-cell penetrating cN50 peptide at molarequivalent (20 μg/g). cN50 peptide, contains the cyclized NLS but lacksthe membrane translocating motif. All injections were delivered in avolume of 100 μl of sterile saline

Immunohistochemistry

Freshly harvested pancreata were fixed in 4% paraformaldehyde-0.1 M PBS(12.07 g of Na₂HPO₄ (dibasic), 2.04 g of KH₂PO₄ (monobasic), 8.0 g ofNaCl, 2.0 g of KCl; pH 7.5, same-day preparation) for 1.5 h at 4° C.under mild agitation, followed by four washings in 0.1 M PBS over aperiod of 2 h at 4° C. under mild agitation. Tissue was equilibrated in30% sucrose in 1×PBS (Invitrogen) overnight at 4° C. until tissuesettled to the bottom of the tube. Pancreata were then frozen in OCT(Sakura Finetek), and cut into 8 μm sections using a cryostat microtome(Leica). Sections were rehydrated with PBS for 2 min before blocking for30 min at room temperature in blocking buffer (5% normal goat serum and1% BSA in 1×PBS), then stained with anti-B220-FITC and anti-CD3 PE for 1h at room temperature, washed with PBS, and mounted in fluorescentmounting medium (Dako). Slides were examined by conventionalfluorescence microscopy using an Olympus BX60 epifluorescencemicroscope. Images were captured using a charge-coupled device cameraand MagnaFire software (Optronics) and optimized for signal-to-noiseusing Adobe Photoshop software (Adobe Systems). A pancreas was deemedinsulitis-free if at least 40 islets were identified and no islet hadevidence of T or B-cell staining.

Flow Cytometry

One million splenocytes were suspended in PBS containing 0.1% azide and3% FCS and surface stained with the following mAbs: RM4-5 (anti-CD4),1D3 (anti-CD19), RA3-6B2 (anti-B220), 53-6.7 (anti-CD8), or 16-10A1(anti-CD80), each conjugated to FITC, PE, PE-Cy7, Cychrome or APC asappropriate Annexin V staining was performed in the appropriate bindingbuffer as supplied. All reagents were obtained from BD Pharmingen (SanJose, Calif.). All samples were analyzed on FACSCalibur flow cytometer(Becton Dickinson, Mountain View, Calif.) using CellQuest software.

CFSE Labeling

Spleens were harvested, and cells were labeled and prepared. Mitoticevents were determined based on the two-fold decrease in fluorescenceintensity with each division of labeled cells.

In Vivo Tracking of Islet-Reactive CD4 T Cell Elimination During NuclearTransport Modifier Treatment

Splenocytes were isolated from NOD BDC2.5 TCR transgenic mice andlabeled with CFSE. A total of 20×10⁶ of these CFSE-labeled cells wasinjected intraperitoneally into NOD-Thy1.1 mice. For BDC2.5 T celltransfers, optimal activation occurs at about 90 h, and so recipientcells were harvested at this time point from pancreatic lymph nodes.Single cell suspensions were prepared and stained with anti-Thy-1.1PE-Cy7 (OX-7), anti-TCR Vβ4-PE (the BDC2.5 transgene utilizes Vβ4 TCR)and anti-CD4 APC (RM4-5) to allow for the identification of thetransferred CD4 T cells using flow cytometry. To allow continuous andfacile peptide delivery throughout the 4-day incubation period, micewere implanted with osmotic pumps the day prior to cell transfer.Implantation was achieved following induction of anesthesia withketamine and xylazine per standard protocol and the pump was placed intoa subscapular location. The wound was closed with a single staple. These4-day pumps deliver 100 μl of fluid at 1 μl/hr and generally begininfusion about 12 hrs after implantation (Alzet, Cupertino, Calif.).

Cytokine Detection by Cytokine Bead Array

Serum or plasma samples were obtained daily by saphenous vein bleeding.Other samples for cytokine determination were obtained from supernatantsof stimulated lymphocyte cultures. Cytokine concentration was determinedby cytokine bead array and comparison to a standard curve (BDBiosciences, San Jose, Calif.) or with the MILLIPLEX mousecytokine-chemokine kit (Millipore, St. Charles, Mo.) according to themanufacturer's specifications.

Clinical Chemistry

ALT and BUN levels were determined on a VITROS 250 Chemistry analyzerusing multi-layered vitros slides and colorimetric or enzyme rate testsas appropriate.

Statistical Analysis

Statistical comparison between groups was performed by log-rankanalysis, Student's t-test or ANOVA as appropriate. A p-value less than0.05 was considered significant.

Example 2

Primary lymphocytes and macrophages from NOD mice are susceptible toattenuation of T cell receptor (TCR)- and Toll-like receptor(TLR)-evoked pro-inflammatory signaling, respectively, by a NuclearTransport Modifier. NOD mice, a widely used model of human T1D,demonstrate genetic predisposition toward autoimmune diabetes asevidenced by numerous immunologic abnormalities of innate and adaptiveimmunity as compared to normal strains of mice. The Nuclear TransportModifier, cSN50 peptide, attenuates TCR- and TLR-evokedcytokine/chemokine production in murine models of acute inflammation andapoptosis using normal C57BL/6 and BALB/c strains (Lin et al (1995)Inhibition of nuclear translocation of transcription factor NF-kappa Bby a synthetic peptide containing a cell membrane-permeable motif andnuclear localization sequence. J Biol Chem 270: 14255-14258; Hawiger J(1999) Current Opinion in Chem. Biology 3:89-94; Liu et al. (2000) JBiol Chem 275: 16774-16778; Liu et al. (2004) J Biol Chem 279:48434-48442). Therefore, it was examined whether agonist-stimulated Tand B lymphocytes and macrophages derived from autoimmune diabetes-proneNOD mice are suppressed by targeting nuclear import withcell-penetrating cSN50 peptide (FIGS. 1A-1C).

The Nuclear Transport Modifier cSN50 suppresses both T Cell Receptor-and Toll-Like Receptor-evoked signaling. Splenocytes from 10 week oldNOD females were isolated and stimulated with anti-CD3/CD28 (2 μg/ml),concanavalin (1 μg/ml) (conA), or LPS (5 μg/ml) in the presence orabsence of Nuclear Transport Modifier (cSN50 peptide at 30 μM).Supernatants were harvested at 72 h and analyzed. The presence of IFN-γas a measure of T cell activation using cytokine bead array was analyzed(**p<0.01 vs. cSN50, t-test) (FIG. 1A). Up-regulation of CD80 (B7.1) asa measure of B cell responsiveness was assessed by flow cytometry(**p<0.01, Student's t-test) on cells co-expressing CD19 as a B cellmarker. (FIG. 1B). Pro-inflammatory cytokine production in bone marrowderived-macrophages cultured in L-conditioned media was examined.Differentiated cells were stimulated with LPS (10 ng/ml) in the presenceor absence of cSN50 (30 μM). (FIG. 1C). cSN50 inhibited production ofTNF-α, IL-1α, and IL-1β (*p<0.05, Student's t-test). Data isrepresentative of three or more experiments.

Isolated NOD splenocytes were stimulated with the T cell agonistsanti-CD3/CD28 or concanavalin A (Con A) in the presence or absence ofthe Nuclear Transport Modifier peptide cSN50 and were measured for theproduction of the pro-apoptotic cytokine IFN-γ. cSN50 suppressed therobust production of this islet-toxic cytokine (FIG. 1A).

The effect of cSN50 on B cells and bone marrow-derived macrophages(BMDM) prepared from NOD mice and stimulated with the pro-inflammatoryagonist LPS that is recognized by Toll-like receptor (TLR) 4 onmacrophages and B cells was assessed. As NOD-derived B lymphocytes arehyper-responsive to LPS in terms of CD80 (B7.1) expression, it wasdemonstrated that this response is attenuated by cSN50 (FIG. 1B).

Expression of islet-toxic cytokines TNF-α, IL-1α, and IL-1β in BMDMobtained from NOD mice was suppressed. Thus, a Nuclear TransportModifier attenuates production of islet-toxic, pro-apoptotic mediatorsevoked by TCR and TLR agonists in primary immune cells derived from NODmice (FIG. 1C).

Example 3

Intracellular delivery of a Nuclear Transport Modifier to the pancreasreduces islet inflammation (insulitis). The highly effective suppressionof pro-inflammatory and pro-apoptotic cytokines in ex vivo analysis ofprimary, NOD-derived immune cells following treatment with cSN50 peptideencouraged in vivo study of Type 1 diabetes in NOD mice. It was firstdetermined whether cSN50 would be delivered to the pancreas, the primarysite of autoimmune attack against beta cells in pancreatic islets. Itwas demonstrated that this cell-penetrating Nuclear Transport Modifierpeptide is delivered to blood leukocytes/lymphocytes, spleen, liver, andlung to suppress acute liver and lung inflammation (Lin et al. (1995) JBiol Chem 270: 14255-14258; Liu et al. (2000) J Biol Chem 275:16774-16778; Liu et al. (2009) Mol Ther 17: 796-802). Using confocalmicroscopy, the pancreatic delivery of FITC-labeled cSN50 peptidefollowing a single intraperitoneal (i.p.) injection was assessed. Rapidand uniform intracellular delivery of FITC-cSN50 was apparent throughoutthe pancreas within 2 h after injection (FIG. 2A).

In vivo intracellular delivery of a Nuclear Transport Modifier to thepancreas attenuates insulitis. cSN50 was conjugated to FITC per themanufacturer's instruction. Animals received one i.p. injection ofFITC-cSN50 or an amount of FITC of equivalent relative fluorescence.Animals were euthanized after 2 h and 10 μm frozen sections were cut andassessed by confocal microscopy. The cSN50-FITC peptide is distributedthroughout the pancreas. In contrast, FITC alone did not penetrate theorgan and only the autofluorescent tissue border is seen. Pancreas isshown at 40× magnification. (FIG. 2A).

The effect of cSN50 delivery on the ongoing insulitis was investigated.The well-characterized accelerated model of autoimmune diabetes in theNOD mouse following a single bolus of cyclophosphamide (Cy), whichsynchronizes progression of T1D, was selected. In this animal model ofhuman Type 1 diabetes, two to four days following Cy injection a peakpro-inflammatory cytokine response is reported, which is followed bydevelopment of overt autoimmune diabetes in 2-4 weeks. Therefore,10-week old female NOD mice received a single injection of Cy in twogroups of seven. Treatment with cSN50 or control (either a non-cellpenetrating peptide denoted cN50 or saline) was initiated 45 h later. Toassure a steady level of Nuclear Transport Modifier in blood andpancreas (see FIG. 2A), a high intensity treatment protocol was adopted.35 μg/g of cSN50 was administered i.p. every two hours for the next 24h. The mice receiving the control peptide received a molar equivalent.Pancreatic sections were analyzed by immunohistochemistry (FIGS. 2B-D).Strikingly, 57% of treated mice following one-day therapy with cSN50were free of insulitis while significant insulitis remained in allage-matched controls receiving Cy alone (p<0.05 vs. treated). Thecontrol finding compares well with other reports of insulitis at thisage which find nearly 100% of NOD mice to demonstrate insulitisaffecting a significant number of pancreatic islets.

cSN50 therapy attenuates ongoing insulitis. (FIGS. 2B, 2C, and 2D). 10week-old NOD females received one injection of Cy (0.2 mg/g). 45 h afterthis injection, treatment was begun with cSN50 (35 μg/g every two hours)and continued for 24 h at which time the seven animals in each group(treatment and control) were euthanized and pancreata obtained. Sectionswere stained for CD3-PE (FIG. 2B) and B220 (FIG. 2C). The differentialinterference contrast (DIC or Nomarski) image is shown in FIG. 2D. Of 7animals assessed in each group, four NOD mice receiving cSN50 werecompletely insulitis free as compared to persistent insulitis in allcontrol group animals (p<0.05, chi-square).

All NOD mice receiving histologic evaluation in cSN50-treated andcontrol groups (n=7 for each) were assigned insulitis scores based onthe severity of inflammation (0=no invading cells, 1=peri-insulits,2=invasive insulitis, 3=minimal residual islet tissue). Comparison ofcontrol to cSN50-treated animals shows significantly more unaffectedislets in treated mice (*p<0.05) and significantly fewer severelyaffected islets (**p<0.001). (FIG. 2E). Mean insulitis score was alsodetermined and showed a value of 0.49 in the cSN50-treated group and1.81 in the control group (p=0.001, Mann-Whitney U test).

One year after the short-course treatment, three treated mice were alsoanalyzed histologically for insulin production and insulitis. Sectionswere stained with a combination of anti-insulin FITC and a combinationof anti-CD3 and anti-B220PE. (FIG. 2F). In long-term survivors (right),insulin staining is detected but insulitis was not observed (FIG. 2F).As a staining control, islets from 15 week old control NODs showsignificant insulitis. Thus, a short-term intracellular delivery ofcSN50 to the pancreas is followed by a rapid reversal of ongoinginsulitis that is otherwise exacerbated in untreated NOD mice when T1Dis synchronized with Cy.

Example 4

Islet-reactive T cells are reduced and AICD is enhanced by intracellulardelivery of a Nuclear Transport Modifier. Ongoing insulitis, as ahallmark of T1D in NOD mice, is driven in part by the persistence ofactivated, autoreactive T and B lymphocytes. They are resistant to AICD,a critical mechanism for the loss of peripheral T cell tolerance. T- andB-lymphocytes were eliminated in the islets of cSN50-treated NOD mice(FIGS. 2B and 2C). Therefore, intracellularly delivered cSN50 mayenhance lymphocyte sensitivity to AICD, thereby reversing the knownresistance of NOD lymphocytes to this process. Changes in totallymphocyte numbers above the transient cell loss following Cy exposurewere not observed (data not shown), however autoreactive cells may havebeen depleted following intense short-term treatment with the NuclearTransport Modifier.

Adoptive transfer of islet-reactive BDC2.5 lymphocytes was utilized totest their persistence and expansion in pancreatic lymph nodes duringtreatment with the Nuclear Transport Modifier. Autoreactive T cellsdisappeared following in vivo treatment with a Nuclear TransportModifier. Donor cells were labeled with CFSE and transferred torecipients that received cSN50 or saline continuously via osmotic pumpfor 4 days (FIG. 3A). Twenty million CFSE-labeled BDC2.5 splenocyteswere transferred to pre-diabetic NOD recipients who received cSN50peptide or saline by osmotic pump. (FIG. 3A). On day 4 post-transfer,pancreatic lymph node cells were harvested and the proliferation profileof CD4+Thy1.1-cells (FIG. 3B) and absolute number of CFSE+Vβ4+ cells(FIG. 3C) within the CD4 compartment was determined. Figures arerepresentative of three separate experiments. Analysis of the pancreaticlymph node demonstrated a striking reduction in transferredislet-reactive cells in the cSN50-treated mice, as demonstrated by asignificant decrease in the absolute number of CFSE-labeled, Vβ4+ cells(*p<0.05, student's t-test) recovered at the end of treatment.Islet-reactive BDC2.5 lymphocytes were precipitously reduced followingcSN50 peptide treatment either by an effect on cell proliferation,lymphocyte survival, or lymphocyte entry to the node.

The effect of the Nuclear Transport Modifier on cell survival andproliferation was assessed by analyzing the sensitivity of NODlymphocytes to AICD in an ex vivo assay. Splenocytes were harvested from10 week old NOD females and stimulated with a defined concentration ofanti-CD3/CD28 for 65 h in the presence or absence of cSN50 beforedetection of apoptosis with annexin-V staining (FIG. 4A). The focus wason the detection of apoptosis in NOD lymphocytes with definitiveevidence of activation as determined by the achievement of at least onecell division.

Activation-induced cell death (AICD) is enhanced following ex vivointracellular delivery of a Nuclear Transport Modifier to splenocytes.Splenocytes from 10 week-old NOD females were labeled with CFSE andstimulated with anti-CD3 (0.05 μg/ml or 2 μg/ml) and anti-CD28 (1 μg/ml)in the presence or absence of cSN50 (30 μM). After 65 h of culture,samples were harvested and labeled with annexin V-PE, anti-CD8 PerCP,and anti-CD4 APC. For analysis of AICD, activation was defined as theachievement of at least one round of division as determined byCFSE-dilution; a representative gating strategy is shown in (FIG. 4A);the undivided peak is highlighted and derived from unstimulated cells inthe same experiment.

Use of CFSE to calculate the number of mitotic events followinganti-CD3/CD28 stimulation revealed that cSN50 had no effect on CD4 orCD8 T cell proliferation (FIG. 4B). The CFSE division history,represented graphically as the number of mitoses per 10,000 CD4 or CD8 Tcells, demonstrated no change in proliferation in CD4 or CD8 T cells inthe presence of cSN50 (p=NS) (FIG. 4B).

In contrast, intracellular delivery of cSN50 increased the sensitivityto AICD in CD3/CD28-stimulated CD4+ and CD8+ T cells (FIG. 4C). Furtheranalysis revealed increased annexin-V staining on activated cells,indicative of cellular apoptosis, for cSN50-treated CD4 and CD8 T cells(p<0.005, paired student's t-test). Data is representative of at least 4separate experiments (FIG. 4C).

As anticipated, CD4 T cells from NOD mice were generally more resistantto AICD but following cSN50 peptide delivery, they displayed asignificant increase in sensitivity to AICD; in fact, the percentage ofCD4 T cells undergoing AICD reached values comparable to T cellsobtained from the non-diabetes prone C57BL/6 strain (FIG. 5A). NODsplenocytes when stimulated with anti-CD3/28 showed reduced apoptosis ascompared to C57BL/6 splenocytes as previously published (*p<0.01,t-test) (FIG. 5A). Addition of cSN50 normalized apoptosis to C57BL/6levels (p=NS). Unstimulated lymphocytes showed high levels of apoptosis,as expected for unactivated lymphocytes in culture; this process was notenhanced by cSN50.

In parallel to the significant change in sensitivity to AICD noted inNOD mice-derived T cells, B lymphocytes also had an increase insensitivity to apoptosis induced by their agonist in the presence ofcSN50 peptide. When B cells were analyzed with similar methods followingstimulation with the TLR4 agonist, LPS, they displayed enhancedpropensity for cell death (p<0.005, paired Student's t-test) (FIG. 5B).Thus, the cSN50 not only reduced the number of islet-reactive T cellsbut restored sensitivity to activation-induced cell death (apoptosis) inNOD CD4 T cells and enhances B cell apoptosis in response to LPS.Splenocytes from 10-week-old NOD or C57BL/6 female mice were isolatedand stimulated with anti-CD3/28 (2 μg/ml) or 5 μg/ml LPS in the presenceor absence of cSN50 (30 μM). After 70 h culture, cells were harvestedand apoptosis was detected by annexin V PE staining with lymphocyteco-labeling with either anti-CD4 APC (FIG. 5A) or anti-CD19 APC (FIG.5B).

Example 5

Immunomodulatory Cytokines IL-5 and IL-10 are enhanced in animalstreated with a Nuclear Transport Modifier. Although cSN50 treatmenteliminated the majority of islet-reactive lymphocytes and facilitatedreduction of insulitis, these islet-reactive and invading immune cellsdid not completely vanish following intense 1- or 4-day delivery ofcSN50 peptide via i.p. injection or osmotic pump, respectively.Therefore, it was considered whether there had been augmentation inregulatory cells or whether the remaining lymphocytes demonstratealteration in their cytokine expression profile. Analysis of theabsolute number of regulatory T cells was determined by intracellularFoxp3 staining and showed no difference between treated and control miceat either the end of treatment (48 h) or two weeks after treatmentconcluded. With respect to cytokine expression, the systemic effect ofcSN50 on cytokines/chemokine production in blood of NOD mice wasmonitored daily in plasma samples in treated and control mice beginningon the day of Cy administration until 12 days following peptidetreatment (day 14 post-Cy bolus). During this period of administration,increased circulating levels of IL-5 were detected on day 2 after Cyadministration (FIG. 6A). No differences were detected in plasma levelsof TNF-α, IFN-γ, IL-10, IL-12, IL-2, or IL-4, Eotaxin, GM-CSF, IL-1α,IL-1β, M-CSF, IL-3, IL-7, IL-9, IL-12 (p40), IL-12 (p70), IL-13, IL-15,IL-17, IP-10, MIP-2, KC, LIF, LIX, MIP-1α, MIP-1β, MIG, RANTES, or VEGF(data not shown) and most of these markers of inflammation were barelydetectable in plasma or were below the limit of detection.

Expression of immunomodulatory cytokines IL-5 and IL-10 is modifiedduring treatment with a Nuclear Transport Modifier. Blood samples wereobtained daily by saphenous vein bleeding beginning on the day of Cychallenge. Cytokine levels in plasma were determined by cytokine beadarray and comparison to a standard curve. cSN50-treated mice demonstrateincreased IL-5 (p<0.05 ANOVA; subsequent analysis of individual days byStudent's t-test shows *p<0.05 for days 2 and 3) during therapy (FIG.6A).

It was considered that the capacity for production of anti-inflammatorycytokines may have been modified although increased systemic productionof anti-inflammatory cytokines, such as IL-10, was not detected.Therefore, splenocytes from NOD females that had received 24-h peptidetherapy beginning two days after Cy acceleration were obtained toexamine the effect of cSN50 peptide on ex vivo cytokine production byimmune cells. Splenocytes from 10 week-old NOD females that had receivedcyclophosphamide were harvested after 24 hr of treatment with cSN50 orcontrol non-cell-penetrating peptide (cN50) and restimulated with LPS toassess their cytokine profile. Supernatants were harvested at 72 h andanalyzed by cytokine bead array for presence of IL-10. Splenocytes fromcSN50 treated animals produced increased levels IL-10 (*p<0.05) (FIG.6B). Data are from three separate experiments. Restimulated splenocytesfrom treated animals showed increased production of theanti-inflammatory cytokine IL-10 suggesting that cSN50 peptide was notsimply a global suppressant of pro-inflammatory cytokine/chemokineproduction in NOD mice but rather a modulator of lymphocyte function inlymphoid organs (FIG. 6B).

Together with results depicted in FIG. 1, these cumulative data indicatethe ability of a Nuclear Transport Modifier to exert a bimodal effect onprimary NOD immune cells by suppressing the expression ofpro-inflammatory mediators, including islet-toxic cytokines like IFN-γand TNF-α while preserving or enhancing anti-inflammatory cytokines,such as IL-10 and Transforming Growth Factor beta, that are likelyassociated with recovery of regulatory T cells (Tregs) followingtreatment with NTM. This effect is likely to contribute to therestoration of peripheral tolerance to autoantigens.

Example 6

Short-term intracellular delivery of a Nuclear Transport Modifier invivo protects NOD mice from autoimmune diabetes for over one year (FIG.7). A striking effect of in vivo delivery of cSN50 peptide on reversalof insulitis through reduction of islet-reactive T and B cells wasestablished. A pre-clinical study of cSN50 peptide in terms of itseffect on T1D progression was performed.

Ten week old female NOD mice received one dose of Cy (0.2 mg/g) tosynchronize autoimmune diabetes progression. After 45 h, a highintensity treatment protocol was utilized to assure a steady level ofNuclear Transport Modifier in blood and pancreas; control mice receivedsaline or control peptide at molar equivalent. Intracellular peptidedelivery was initiated with cSN50 (35 μg/g) or with control andcontinued every 2 h for 48 h. Blood glucose was assessed twice weekly.cSN50-treated mice (n=20) were significantly protected from diabetesprogression as compared to saline-treated control (n=10, p<0.0001) orthe non-cell-penetrating peptide cN50-treated control (n=10, p=0.006). Acomparison of cSN50-treated vs. the combined control groups asillustrated in FIG. 7 also demonstrated significant protection(p=0.0002, log-rank test).

As documented in FIG. 7, the majority of NOD mice (90%) were rendereddiabetes-free following this short-term treatment of only 2 days withcSN50. In contrast, 90% of control mice developed diabetes by 100 daysafter Cy bolus. The progression to diabetes in control group proceededin two phases. On days 10-30, a first wave of diabetes developed andcSN50 afforded 100% protection against this process. In the second wave,corresponding closely to the normal progression of spontaneous diabetesbetween days 60-100 post-Cy (ages 18-23 weeks), protection wasincomplete but still substantial even though more than two months hadelapsed since the last dose of Nuclear Transport Modifier. Strikingly,the cSN50 peptide-treated mice remained euglycemic, which obviated theneed for insulin replacement therapy. Importantly, they did not displayovert signs of toxicity during 365 days of observation. The lack oftoxicity of intensive treatment in this model was supported by normalweight gain, no signs of acquired infection, and normal clinicalchemistries (ALT and BUN levels were not significantly different fromcontrol, FIGS. 8A and 8B).

Short-term treatment of NOD mice with a Nuclear Transport Modifier showsno effect on measured clinical biomarkers of liver and kidney toxicity.Ten week old female NOD mice received one dose of Cy (0.2 mg/g); after45 h, intracellular peptide delivery was initiated with cSN50 (35 μg/g)or with control cN50 and continued every 2 h for 48 h. Serum wasobtained daily from the saphenous vein and measurements of ALT (FIG. 8A)and BUN (FIG. 8B) were performed in 7 mice in each group. No significantdifferences were determined. Measurements for alkaline phosphatase,creatinine, creatine kinase, and total bilirubin were below the limit ofdetection in both groups.

Moreover, anti-cSN50 peptide antibody induction in an ELISA assay wasnot detected where the limit of detection based on a titration ofcSN50-reactive IgG antibody was 10 ng/mL (O.D. values for serum were notsignificantly different from secondary antibody alone).

Thus, short-term targeting of the nuclear import of stress-responsivetranscription factors with cSN50 peptide suppressed acceleratedautoimmune diabetes progression and rendered thriving NOD mice free ofdiabetes progression for at least one year.

Example 7

Islet cell transplantation will be performed in order to provide insulinproducing capability to an individual with Type 1 Diabetes whose betacells within the pancreatic islets no longer produce insulin. In islettransplantation, islets are taken from the pancreas of a deceased donor.Typically, a subject will receive 10,000 islet equivalents per kilogramof body weight. After transplantation, the beta cells in the islets willbegin to make and release insulin. The subject may require twotransplants to achieve insulin independence. Donor antigen presentingcells may be removed from the islets before transplantation (Huang etal., (2008) Endocrine Reviews 29(5): 603-630). The beta cells of theislets will be stored and transported in a composition comprising cSN50or cSN50.1 after removal from the donor and before implantation into therecipient. The composition comprising cSN50 or cSN50.1 may be added tothe islets during the isolation process from the donor.

Example 8

Islet cell transplantation will be performed in order to provide insulinproducing capability to an individual with Type 1 Diabetes whose betacells within the pancreatic islets no longer produce insulin. In islettransplantation, islets are taken from the pancreas of a deceased donor.Typically, a subject will receive 10,000 islet equivalents per kilogramof body weight. After transplantation via portal vein into liver, thebeta cells in the islets will begin to make and release insulin. Thesubject may require two transplants to achieve insulin independence. Acomposition comprising cSN50 and cSN50.1 may be administered to therecipient before and after islet transplantation. Treatments with cSN50and cSN50.1 may be administered during treatment with growth factors(Huang et al., (2008) Endocrine Reviews 29(5): 603-630). Variations andmodifications to the preferred embodiments of the invention describedherein will be apparent to those skilled in the art. It is intended thatsuch variations and modifications may be made without departing from thescope of the invention and without diminishing its attendant advantages.

Other Embodiments

Any improvement may be made in part or all of the compositions, kits,cells, and method steps. All references, including publications, patentapplications, and patents, cited herein are hereby incorporated byreference. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended to illuminate the invention anddoes not pose a limitation on the scope of the invention unlessotherwise claimed. Any statement herein as to the nature or benefits ofthe invention or of the preferred embodiments is not intended to belimiting, and the appended claims should not be deemed to be limited bysuch statements. More generally, no language in the specification shouldbe construed as indicating any non-claimed element as being essential tothe practice of the invention. This invention includes all modificationsand equivalents of the subject matter recited in the claims appendedhereto as permitted by applicable law. Moreover, any combination of theabove-described elements in all possible variations thereof isencompassed by the invention unless otherwise indicated herein orotherwise clearly contraindicated by context.

What is claimed is:
 1. A method of preserving viability ofinsulin-producing beta cells in pancreatic islets in a mammalian subjecthaving diabetes or prone to developing diabetes comprising administeringa composition comprising a Nuclear Transport Modifier to the mammaliansubject in an amount sufficient to reduce accumulation of autoreactivelymphocytes in the pancreatic islets and pancreatic lymph nodes andenhance cell death of autoreactive lymphocytes in the pancreatic islets,wherein insulin producing capacity of the insulin-producing beta cellsin the pancreatic islets is preserved, and wherein the Nuclear TransportModifier is cSNS0.1 having the amino acid sequence set forth in SEQ IDNO:2.
 2. The method of claim 1, wherein the composition is in an amountsufficient to enhance expression of at least one anti-inflammatorycytokine in the pancreatic islets.
 3. The method of claim 2, wherein theat least one anti-inflammatory cytokine is IL-10.
 4. The method of claim1, wherein administration of the composition decreases autoimmuneinflammation by attenuating expression of at least one stress-responsivetranscription factor-regulated gene.
 5. The method of claim 1, whereinautoimmune inflammation-induced apoptosis of the insulin-producing betacells is decreased or prevented.
 6. The method of claim 1, whereinsensitivity to activation-induced cell death in T and B lymphocytes thatinfiltrate pancreatic islets is restored.
 7. The method of claim 1,wherein the mammalian subject is genetically prone to develop Type 1diabetes and the composition is administered to the mammalian subjectbefore detection of hyperglycemia or after detection of hyperglycemia.8. The method of claim 1, further comprising monitoring the subject fordevelopment of diabetes after administering the composition comprisingsaid Nuclear Transport Modifier to the mammalian subject.
 9. The methodof claim 1, further comprising the step of measuring blood glucoselevels in the mammalian subject after administering the compositioncomprising said Nuclear Transport Modifier to the mammalian subject. 10.A method of preserving viability of insulin-producing beta cells inpancreatic islets in a mammalian subject having diabetes or prone todeveloping diabetes comprising: administering a composition comprising aNuclear Transport Modifier to the mammalian subject, wherein insulinproducing capacity of the insulin-producing beta cells in the pancreaticislets is preserved, wherein the composition is administered to themammalian subject at at least one time point selected from the groupconsisting of: prior to the mammalian subject receiving a transplant ofinsulin-producing beta cells, concomitant with the mammalian subjectreceiving a transplant of insulin-producing beta cells, and subsequentto the mammalian subject receiving a transplant of insulin-producingbeta cells; and transplanting insulin-producing beta cells into themammalian subject, wherein the Nuclear Transport Modifier is cSN50.1having the amino acid sequence set forth in SEQ ID NO:2.
 11. A method oftreating diabetes in a mammalian subject comprising administering acomposition comprising a Nuclear Transport Modifier to a mammaliansubject having diabetes in an amount sufficient for preservinginsulin-producing capacity of beta cells in the mammalian subject'spancreas and for at least one of: reducing accumulation of autoreactivelymphocytes in the subject's pancreas; enhancing expression ofanti-inflammatory cytokines in the subject's pancreas; and restoringtolerance to pancreatic islets autoantigens, wherein administration ofthe composition results in remission of the diabetes in the mammaliansubject, and wherein the Nuclear Transport Modifier is cSN50.1 havingthe amino acid sequence set forth in SEQ ID NO:2.
 12. The method ofclaim 11, wherein the composition is delivered to the mammaliansubject's pancreas.
 13. The method of claim 11, wherein the mammaliansubject has type 1 diabetes, and the remission occurs in the presence ofinsulin therapy.
 14. The method of claim 11, wherein the mammaliansubject has type 1 diabetes, and the remission occurs in the absence ofinsulin therapy.
 15. The method of claim 11, wherein the composition isin an amount sufficient to enhance expression of at least oneanti-inflammatory cytokine in the pancreatic islets.
 16. The method ofclaim 15, wherein the at least one anti-inflammatory cytokine is IL-10.17. The method of claim 11, wherein administration of the compositiondecreases autoimmune inflammation by attenuating expression of at leastone stress-responsive transcription factor-regulated gene.
 18. Themethod of claim 11, wherein autoimmune inflammation-induced apoptosis ofthe insulin-producing beta cells is decreased or prevented.
 19. Themethod of claim 11, wherein sensitivity to activation-induced cell deathin T and B lymphocytes that infiltrate pancreatic islets is restored.20. The method of claim 11, wherein the composition is administered tothe mammalian subject at at least one time point selected from the groupconsisting of: prior to the mammalian subject receiving a transplant ofinsulin-producing beta cells, concomitant with the mammalian subjectreceiving a transplant of insulin-producing beta cells, and subsequentto the mammalian subject receiving a transplant of insulin-producingbeta cells.
 21. The method of claim 11, further comprising the step ofmeasuring blood glucose levels in the mammalian subject afteradministering the composition comprising said Nuclear Transport Modifierto the mammalian subject.
 22. A method of treating diabetes in amammalian subject comprising administering a composition comprising aNuclear Transport Modifier to a mammalian subject having diabetes in anamount sufficient for preserving insulin-producing capacity of betacells in the mammalian subject's pancreas when administered incombination with insulin and for at least one of: reducing accumulationof autoreactive lymphocytes in the subject's pancreas; enhancingexpression of anti-inflammatory cytokines in the subject's pancreas; andrestoring tolerance to pancreatic islets autoantigens, whereinadministration of the composition in combination with the insulinresults in remission of the diabetes in the mammalian subject, andwherein the Nuclear Transport Modifier is cSN50.1 having the amino acidsequence set forth in SEQ ID NO:2.
 23. The method of claim 22, whereinthe composition is delivered to the mammalian subject's pancreas. 24.The method of claim 22, wherein the composition is in an amountsufficient to enhance expression of at least one anti-inflammatorycytokine in the pancreatic islets.
 25. The method of claim 24, whereinthe at least one anti-inflammatory cytokine is IL-10.
 26. The method ofclaim 22, wherein administration of the composition decreases autoimmuneinflammation by attenuating expression of at least one stress-responsivetranscription factor-regulated gene.
 27. The method of claim 22, whereinautoimmune inflammation-induced apoptosis of the insulin-producing betacells is decreased or prevented.
 28. The method of claim 22, whereinsensitivity to activation-induced cell death in T and B lymphocytes thatinfiltrate pancreatic islets is restored.
 29. The method of claim 22,wherein the composition is administered to the mammalian subject at atleast one time point selected from the group consisting of: prior to themammalian subject receiving a transplant of insulin-producing betacells, concomitant with the mammalian subject receiving a transplant ofinsulin-producing beta cells, and subsequent to the mammalian subjectreceiving a transplant of insulin-producing beta cells.
 30. The methodof claim 22, further comprising the step of measuring blood glucoselevels in the mammalian subject after administering the composition incombination with the insulin to the mammalian subject.
 31. A method ofpreserving insulin-producing beta cells comprising delivering acomposition comprising a Nuclear Transport Modifier to a population ofinsulin-producing beta cells in an amount sufficient to preserveviability of the insulin-producing beta cells and the insulin-producingcapacity of the insulin-producing beta cells if the insulin-producingbeta cells are subsequently exposed to proinflammatory stimuli, whereinthe Nuclear Transport Modifier is cSN50.1 having the amino acid sequenceset forth in SEQ ID NO:2.
 32. The method of claim 31, wherein thepopulation of insulin-producing beta cells is to be transplanted into amammalian subject in need thereof, and the composition is delivered tothe population of insulin-producing beta cells prior to transplantation.33. The method of claim 32, wherein a pancreas comprising the populationof insulin-producing cells is transplanted into the mammalian subject,and the composition is in an amount sufficient to reduce accumulation ofautoreactive lymphocytes in the pancreatic islets and pancreatic lymphnodes and enhance cell death of autoreactive lymphocytes in thepancreatic islets.
 34. The method of claim 33, wherein the compositionis in an amount sufficient to enhance expression of at least oneanti-inflammatory cytokine in the pancreatic islets.
 35. The method ofclaim 34, wherein the at least one anti-inflammatory cytokine is IL-10.36. The method of claim 33, wherein the composition is in an amountsufficient to decrease autoimmune inflammation in the mammalian subjectby attenuating expression of at least one stress-responsivetranscription factor-regulated gene.
 37. The method of claim 34, whereinautoimmune inflammation-induced apoptosis of the population ofinsulin-producing beta cells is decreased or prevented.
 38. A method oftreating Type 2 diabetes in a mammalian subject comprising administeringa composition comprising a Nuclear Transport Modifier to the mammaliansubject having Type 2 diabetes in an amount sufficient for loweringblood glucose levels and reducing insulin resistance, whereinadministration of the composition results in remission of the Type 2diabetes in the mammalian subject, and wherein the Nuclear TransportModifier is cSN50.1 having the amino acid sequence set forth in SEQ IDNO:2.
 39. The method of claim 38, further comprising the step ofmeasuring blood glucose levels in the mammalian subject afteradministering the composition comprising said Nuclear Transport Modifierto the mammalian subject.
 40. A method of preserving viability ofinsulin-producing beta cells in pancreatic islets in a mammalian subjecthaving diabetes or prone to developing diabetes comprising administeringa composition comprising a Nuclear Transport Modifier to the mammaliansubject, wherein the Nuclear Transport Modifier is cSNS0.1 having theamino acid sequence set forth in SEQ ID NO: 2 and insulin producingcapacity of the insulin-producing beta cells in the pancreatic islets ispreserved.